Impact of RNA quality on reference gene expression stability.

Gene expression quantification methods are important tools in the understanding of the molecular events underlying human diseases and in the identification of diagnostic and therapeutic targets. Generally, the messenger RNA (mRNA) used for these analyses is derived from human biopsies obtained after surgery. As a consequence, several steps during tissue handling have to be carefully controlled in order to preserve the quality and integrity of the RNA material. It is well known that RNA is sensitive to degradation by postmortem processes and inadequate sample handling or storage (1). However, RNA integrity control is often not systematically performed prior to (PCR-based) downstream analyses. While in the past, RNA quality could often not be assessed due to the limited availability of the precious sample (e.g., from microdisected cells or small biopsies), the advent of capillary gel electrophoresis and (sample retention) spectrophotometry technologies (e.g., NanoDrop® ND-1000; NanoDrop Technologies, Wilmington, DE, USA ) has addressed this issue, allowing quality estimations using only nanograms (or even picograms) of total RNA (2). In addition, amplification of RNA is now an alternative method to obtain sufficient amounts to conduct gene expression studies when postmortem tissues are scarce; however, assessment of RNA quality based on the 18S and 28S ribosomal RNA bands is often not possible anymore after amplification. Furthermore, it remains to be determined whether the amplified mRNA can faithfully be used to assess RNA quality of the starting material. Apart from RNA quality, the choice of a proper set of reference genes for accurate normalization is another crucial factor with a profound impact on the reliability of the obtained gene expression levels (3). Reference genes are expressed constitutively in every cell; however, their expression can be regulated with diseases state, during cellular proliferation, due to cellular composition and by mitogenic stimuli (e.g., growth factors) (4,5). Furthermore, it is now known that life styles and genetic make-up of individuals can influence mRNA expression (6). That is why the validation of the expression stability of reference genes remains an important step to ensure the accuracy and reliability of gene expression studies. The objective of this study was to analyze the influence of RNA degradation on the stability and expression pattern of different internal control genes. To this purpose, 10 commonly used reference genes were quantified in both intact and degraded RNA from clinical specimens obtained from ethmoidal and maxillary sinuses collected from patients with nasal polyposis (NP) and chronic rhinosinusitis (CRS). Sixteen clinical tissue samples (30 mg) were homogenized in Tri-reagent buffer (Sigma, St. Louis, MO, USA) (1 mL/50–100 mg of tissue) in a chilled pestle mortar. Total RNA isolation and cDNA synthesis were performed as described previously (7). RNA Impact of RNA quality on reference gene expression stability